Starch nanocrystals (SNCs) and their applications in hydrogels

Starch is an abundant natural polymer, produced by various plants as a source of stored energy.¹⁰² The starch structure is multi-scale and lies in the (i) starch granule (2-100 μm), where displays as (ii) growth rings (120-500 nm). The growth rings are comprised of (iv) blocklets (20-50 nm) with (iii) amorphous and crystalline lamellae (9 nm) containing (vii) amylopectin and (viii) amylose chains (0.1-1 nm), as presented in Figure 9a.^103-105 All starches are constituted by α-D-glucopyranose units in linear chains linked by α (1→4) bonds in amylose and in branched chains linked by α (1→6) bonds from the linear chains in amylopectin. They are ordered in alternating crystalline and amorphous lamellae (9 nm) in the 2-100 μm starch granules.

SNCs are generated by acid hydrolysis, enzymatic hydrolysis, where the disordered or less ordered parts of starch granules are preferentially hydrolyzed, the crystalline regions remain.¹⁰⁶ Distinguished from CNCs and ChNCs, SNCs are crystalline platelets. The structure and

21

morphology of the isolated starch nanocrystals commonly depend on the source of starch with various crystallization type, differed relative proportion of crystalline amylose and amylopectin.¹⁰⁷ The size and yield of SNCs are influenced by the hydrolysis conditions (such as acid type,acid concentration, temperature, and time).

Figure 9. Structure of starch and the SNCs extracted from A-crystalline type and B-crystalline type starch granules. (a) Concentric multiscale structure of starch. (i) Typical starch granules from normal maize (30 μm), (ii) Amorphous and semicrystalline growth rings (120-500 nm), (iii) amorphous and crystalline lamellae (9 nm), (iv) magnified details of the semi-crystalline growth ring with blocklets (20-50 nm), constituting unit of the growth rings, (v) amylopectin double helices forming the crystalline lamellae of the blocklets, (vi) nanocrystals: other representation of the crystalline lamellae called SNC when separated by acid hydrolysis, (vii) amylopectin’s molecular structure, (viii) amylose’s molecular structure (0.1-1 nm).

(Reproduced from TANG et al.¹⁰³ Copyright from Elsevier 2006; DONALD et al.¹⁰⁴ Copyright fromScience and Technology Facilities Council 1997; GALLANT et al.¹⁰⁵ Copyright from Elsevier 1997) (b) SNCs extracted from different starch granules of waxy maize starch (A-type) and high amylose starch (B-type). (Reproduced from LECORRE et al.¹⁰⁷ Copyright from Springer 2011)

In recent years, chemical modifications were performed on their hydroxyl groups of SNCs, such as chemical reaction with small molecules, polymer chains grafting onto the surface with coupling agents, polymer chains growing on the surface with polymerization of monomers.¹⁰⁸ Those surface modifications ensure SNCs compatible with many polymer matrices.

22

Figure 10. SNCs composite hydrogel as cell-instructive materials. (Reproduced from PILUSO et al.¹¹⁰ Copyright from American Chemical Society 2019)

Hydrogels with SNCs. SNCs in hydrogels were normally used as mechanical reinforcement fillers with similar improvement mechanism with CNCs and ChNCs. Owing to the unique excellent biocompatibility, low toxicity, biodegradability, they are promising materials in drug carriers, tissue engineering, and skin adhesives.¹⁰⁹ Piluso et al. developed 3D microenvironments with starch nanocrystals embedding in the gelatin matrix as cell-instructive materials. The incorporation of SNCs led to improved compressive modulus, at the same time, composite hydrogel showed potential usage for cartilage tissue engineering with the evidence that the chondrogenic progenitor ATDC5 cells maintained viability around 90% but displayed a round morphology, especially in the hydrogels with SNCs (Figure 10).¹¹⁰

Overall, applying polysaccharide nanocrystals to construct functional hydrogels attracts growing interests both in academia and industry. Within the scope of polysaccharide nanocrystals, CNCs still hold great promise in functionalizing hydrogels in light of their mature synthesis technology with specific surface groups on demand, rigid rod-like morphology and their extraordinary liquid crystal nature.

23 2. Objective of the study

Hydrogels are crosslinked 3D polymer networks with amounts of water in their highly porous structure. The crosslinked polymer networks endow hydrogels with the properties of soft and elastic solid, while the high-water content in hydrogels gives rise to liquid-like properties, such as good permeability to various chemicals, plastic behavior and adjustable optical characteristics. In addition, hydrogels have some unique properties, such as the responsiveness and swelling, brought by their tailorable polymer network and exchangeable aqueous system.

All these features make hydrogels promising semi/solid materials with diverse practical applications.

Great efforts have been made to hydrogel functionalization, especially, functionalization by diverse nanocomposites, to satisfy various applications. As aforementioned in the introduction section, CNCs composite hydrogels have great potential as functional materials owing to their bio-based and renewable nature, excellent mechanical properties, numerous chemical-active groups on the surface, and unique optical properties. However, the CNCs applied in functional hydrogels and CNCs assistance in functional hydrogel fabrication, as well as the transferring of unique optical properties from CNCs to hydrogels and dry materials still need further research.

Therefore, the present study aims to develop functional composite hydrogels based on CNCs.

The main objective covers the following points:

1. To prepare crosslinkable CNCs with surface-attached methyl acrylamide groups, and to use them as reinforcement nanocrosslinkers in the hydrogel actuators. (Publication 1)

2. To prepare closed hollow hydrogels with distinctively tunable inner and outer walls through the assistance of CNCs to improve spatial crosslinking distinction. (Publication 2)

3. To transfer and integrate the birefringence of CNCs and surface plasmon resonance of gold nanorods in fabricating optical polymer films based on the thixotropy of dynamic hydrogels.

(Publication 3)

24 3. Results and discussion

3.1. Bilayer hydrogel actuators with programmable and precisely tunable motions As reported in Publication 1, a novel type of bilayer hydrogel actuators (BHAs) was prepared comprising of a poly(N-isopropylacrylamide) (PNIPAm) and a poly(N-hydroxyethyl acrylamide) (PHEAm) hydrogel layer with various compositions. Cellulose nanocrystals (CNCs) are water dispersible with a modifiable surface. At first, we prepared methyl acrylamide groups modified CNCs (CNCs-MAm) (Figure 11).

Figure 11. Surface modifications of CNCs.

Then, as shown in Figure 12a, BHAs were prepared via a two-step method. The first layer was chemically crosslinked PNIPAm hydrogels as the active layer. Then, various PHEAm layers were fabricated above the PNIPAm layer as the cooperating layers. BHAs were obtained after equilibrated in deionized water. PHEAm hydrogels in diverse BHAs were prepared with three different compositions: bulk crosslinked PHEAm in BHAs/P, PHEAm networks containing reinforcing CNCs (PHEAm/CNCs) in BHAs/CNCs, and PHEAm networks containing crosslinkable CNCs-MAm (PHEAm/CNCs-MAm) in BHAs/CNCs-MAm.

The scanning electron microscopy (SEM) images in Figure 12b clearly showed the distinguishable PHEAm/CNCs-MAm layer and PNIPAm layer with different pore topologies within BHAs after freeze-drying. A much denser area was visible at the interface of the two layers, indicating the interpenetration of two kinds of polymer chains in this interfacial area.

This dense interface in turn enhanced the connection of the two hydrogel layers. As a result, the covalently crosslinked interactions tightly fix the two hydrogel layers together, which were sufficient to tolerate the swelling and actuation behaviors triggered by various stimuli.

25

Furthermore, the introduction of CNCs and crosslinkable CNCs-MAm led to much smaller pore size for the cooperating hydrogel layers.

Figure 12. Schematic illustration for the preparation and the microstructures of BHAs. (TEM image of CNCs-MAm with the scale bar of 50 nm).

The smaller pore size could be attributed to the extra crosslinking brought by the CNCs-MAm, which can be further proved by the smallest swelling ratio for PHEAm/CNCs-MAm comparing with its in PHEAm/CNCs and PHEAm/P (Figure 13a). The mechanical properties of various BHAs with distinct compositions were further characterized. In particular, the mechanical properties of cooperating PHEAm layers based on PHEAm with various compositions were characterized due to their distinct microstructures, while the active PNIPAm layers maintained equal in all BHAs. As exhibited in Figure 13b, the addition of CNCs and CNCs-MAm improved the stiffness and toughness of hydrogels with diverse extents. While CNCs only acted as neutral nanofillers and showed limited enhancement on the breaking stress of resulting hydrogels to about 2.5 times of PHEAm hydrogels, crosslinkable CNCs-MAm efficiently increased the breaking stress of obtained hydrogels to roughly 14 times of PHEAm hydrogels.

Furthermore, the tensile tests clearly displayed the effective toughening by introducing CNCs and CNCs-MAm into the PHEAm hydrogels.

26

Figure 13. Swelling behaviors and mechanical properties of the cooperating layers

Considering the great difference in mechanical properties caused by the introduction of CNCs and CNCs-MAm in cooperating layers, we further studied the thermal behaviors of BHAs containing diverse cooperating layers. PNIPAm has a (Lower critical solution temperature) LCST at about 32 °C and exhibits greater hydrophobicity at the temperature above its LCST.

When BHAs were immersed into water of 40 °C, PNIPAm chains aggregated which induced shrinkage of the PNIPAm layers, all BHAs bended to the PNIPAm side (Figure 14a). The magnitudes of their dynamic bending motions were illustrated by the corresponding curvatures of BHAs after a certain time. Obviously, BHAs containing diverse compositions showed distinct bending amplitudes according to their curvatures after the same bending times during the dynamic process (Figure 14b). Generally, the continuous bending completed within 6 min and the shapes of the BHAs at 6 min were set as the final state. The BHAs/CNCs-MAm exhibited the smallest motion range and the curvature lay between 0.018-0.2, while BHAs/P showed the largest bending range with the curvature roughly between 0.11-0.6. The curvatures of BHAs/CNCs during the bending changed from 0.12 to 0.47. Because of the equal PNIPAm layers, this difference should be mainly attributed to the significant difference in the mechanical properties of the cooperating layers. The stiffer cooperating layer would sacrifice the bending amplitude for the BHAs.

27

Figure 14. Dynamic thermal behaviors of BHAs in water of 40 °C (scale bars: 1 cm).

However, when the BHAs were designed as soft grippers to fulfill the grasping and releasing actions via varying the temperature of DI water (Figure 15a). With the reinforcement of the cooperating PHEAm layers with CNCs or CNCs-MAm, the grabbing capabilities of BHAs were largely increased (Figure 15b). BHAs/CNCs-MAm showed the highest maximum weight ratio and could lift items that were about 18 times the weight of own polymer weight. While the BHAs/CNCs could hold items of roughly 1400 wt% of own polymer weight, and BHAs containing PHEAm only could lift objects of 800 wt% of own polymer weight. Therefore, stronger cooperating layers highly promoted the loading capability of BHAs, independent on the active PNIPAm layers.

Figure 15. BHAs designed as gripper to grasp and release of target object (scale bars: 1 cm).

maximum weight ratios = maximum weights that actuators can lift/own polymer weights.

28

In addition, the thermal triggered actuator would deform until to its final state with uncontrollable intermediate state. These BHAs managed to bend with controllable motion amplitude which can be programmed to stop at a certain amplitude via their unique solvent-responsive properties in ethanol/water mixtures. As shown in Figure 16a, the co-nonsolvency property of PNIPAm in ethanol/water mixtures endows PNIPAm hydrogels with the ability to swell and deswell depending on the ratio of both solvents. At the same time, various PHEAm cooperating layers shrink more strongly with increasing ethanol content (Figure 16b).

Figure 16. Swelling behaviors of PNIPAm layers (a) and various PHEAm layers (b) in diverse water/ethanol mixtures.

Nevertheless, these two different behaviors of the PNIPAm and PHEAm hydrogel layers in the ethanol/water mixtures synergistically contributed to extraordinary solvent-responsive behaviors of our BHAs. Subsequently, this co-solvent actuation not only generated bidirectional bending motions, but also the bending amplitudes (represented by their corresponding curvatures) could be adjusted by changing the solvent composition, afterwards, the BHAs could be fixed at a certain bending state (Figure 17a, b). It is obvious that the compositions and thus the mechanical properties of the cooperating PHEAm layers significantly affected the performance of these diverse BHAs (Figure 17a, b). Thus, this ethanol-triggered actuation allowed BHAs to autonomously change their bending direction and amplitudes according to the ethanol content in the surrounding environment. Moreover, artificial grippers based on such BHAs can be used in complex conditions, such as to transport

29

items through gates (Figure 17c). As showed on the gripper of BHAs/CNCs, the gripper in their smallest size easily passed the narrow gate within the ethanol/water mixture with 20 vol%

ethanol. By increasing the ethanol content to 80 vol%, the gripper expanded and could grab a target object. By diluting the solvent to ethanol content of 20 vol%, the gripper easily grabbed the object and took it through the narrow gate. Eventually, this object was released in another surrounding as designed, e.g. in pure water. Thus, such BHAs showed promising practical applications in diverse fields.

Figure 17. Dynamic behaviors of BHAs triggered by ethanol/water mixture. All BHAs were constituted by the PNIPAm layers on the right side and PHEAm layers on the left side (scale bars: 10 mm).

In the present section, we reported a series of bilayer hydrogel actuators. They demonstrated advantageous controllable thermal-responsive and solvent-driven actuation performance. The incorporation of strengthening CNCs and CNCs-MAm in the cooperating PHEAm layers effectively improved the loading capacities of BHAs, although the they reduced motion amplitude. The ethanol driven actuation provides the feasibility to exactly tune the bending amplitude and bending direction of BHAs by adjusting the ethanol content. Thus, these

pre-30

programmable motions and the feasibility to spontaneously freeze the state of BHAs demonstrate their great peculiarity.

3.2. Temperature-Responsive, Manipulable Cavitary Hydrogel Containers by Macroscopic Spatial Surface-Interior Separation

Synthetic macroscopic materials transforming from bulk solid or semisolid to closed hollow structure with distinct outer and inner microstructures is rarely reported. In Publication 2, we demonstrated an in situ method for directing macroscopic spatial surface-interior separation from bulk dynamic hydrogels to closed 3D Janus hollow hydrogels via constructing competitively crosslinking gradient within dynamic hydrogels. The original crosslinking of phenylboronic acid/catechol complexes was disrupted and replaced by stronger crosslinking of Fe³⁺/catechol associations, generating gradually weakened crosslinking from outside to inside.

Sequential decomposition of weak crosslinking in the inner core within the densely crosslinking hydrogel shell, leading to closed hollow hydrogels with tunable dense outer shell and fluffy inner surface.

Figure 18. Schematic illustration for the preparation of closed hollow hydrogels.

In the present work, firstly, dynamic hydrogels were prepared comprising of PNIPAm hydrogel crosslinked by the dynamic covalent bonds of phenylboronic acid/catechol with the presence of CNCs in buffer solutions of pH 10 (associate constant Ka ≈ 0.919×10³ M^-1) (Figure 18-i).

Then, the dynamic hydrogels were immersed into ferric chloride solution (0.1 M in pH 10

31

borate buffer solution) for controlled time, which allowed the Fe³⁺ to diffuse into hydrogels from the surrounding solution (Figure 18-ii). During this process, the Fe³⁺ ions formed complexes with catechol and replaced initial boronate ester bonds due to their much higher Ka (10³⁷-10⁴⁰ M^-1). Along with the Fe³⁺ penetration, the Fe³⁺ concentration gradient resulted in gradually weakened Fe³⁺/catechol crosslinking in the hydrogel network from surface to core.

After soaking in Fe³⁺ solution with certain time, the treated hydrogels were transferred into DI water to dialyze until the weight was constant (Figure 18-iii). During this process, the hydrogel swelled largely driven by osmotic pressure, resulting in decomposition of weak crosslinking, but strong crosslinking can maintain. As a result, the outer part with strong crosslinking formed the stable hydrogel wall. At the same time, the inner part where the Fe³⁺ did not reach was dominated by boronate ester bonds crosslinked part dissociated into extremely loosely crosslinked polymer chains. As a result, the thin flat bulk hydrogels experienced a macroscopic spatial surface-interior separation process to generate 3D closed continuous hydrogels with enclosed solutions within hollow hydrogels (HHs/P represented polymeric hollow hydrogels without CNCs, and HHs/CNCs with CNCs) (Figure 18-iv).

Figure 19. Formation process of hollow hydrogels with various Fe³⁺ immersing time. Scale bar: 1 cm.

Besides the complex of Fe³⁺/catechol，CNCs also play an important role for the formation of hollow hydrogels. CNCs with numerous carboxyl groups (-COOH) on the surfaces significantly influenced the formation process of hollow hydrogels with respect to various immersion times in Fe³⁺ solution (Figure 19a). The dynamic hydrogels were immersed in Fe³⁺

solution for 1 to 8 min and then immediately transferred to DI water to dialyze. Obvious

32

HHs/CNCs were generated when treated by Fe³⁺ solution for 1 to 7 min, while 8 min treatment would result to bulk solid core hydrogels due to the uniform strong crosslinking in the whole hydrogel. In comparison, without the addition of CNCs, hollow hydrogels were only appeared with 1 to 3 min treatment, while longer immersion time already resulted in bulk solid hydrogels.

This evidence suggested the addition of CNCs could improve the spatial distinction within hydrogels, which can also be explained by the shorter formation time required to equilibrium during the evolution process. As shown in Figure 19b, HHs/CNCs with 3 minutes treatment of Fe³⁺ can reach the equilibrium after about 9 days, whereas, HHs/P needed roughly 20 days to equilibrium. Along with the diffusion rout of Fe³⁺ ions, -COOH on CNCs would complex with Fe³⁺ to provide additional crosslinking comparing with non-composite hydrogels. This extra crosslinking apparently retarded the penetration of Fe³⁺ ions, increasing the spatial crosslinking difference in hollow hydrogel formation.

Other than CNCs, different soaking times in Fe³⁺ solutions allowed steadily control of the wall thicknesses and their outer and inner surface morphologies as well as the microstructures in hollow hydrogels. Together with increasing the immersion time in Fe³⁺ solutions, the shell thickness of freeze-dried hollow hydrogels increased from roughly 0.22 mm to 0.75 mm (Figure 20a). At the same time, as displayed in SEM images, these freeze-dried hollow hydrogels after diverse immersion times in Fe³⁺ solutions had distinct outer and inner layers (Figure 20b). While the outer layers contained porous microstructures as typically for crosslinked networks in hydrogels, the inner layer contained a fluffy mat of loosely crosslinked polymer chains. This is because of different crosslinking densities in the outer and internal layers. Moreover, the pore sizes of outer surfaces in hollow hydrogels were largely decreased from ~9.7 ± 0.4 μm to ~3 ± 0.5 μm with increasing exposure time in Fe³⁺ solutions from 1 to 7 min, indicating further the formation of denser layers with longer crosslinking time. At the same time, the inner polymer mat became denser with longer treatment time in Fe³⁺ solutions and therefore increasing amount of penetrated Fe³⁺ ions. Furthermore, the outer and inner surface of the shells in dried HHs/CNCs displayed different wettabilities, which partly depended on immersion times in Fe³⁺ solutions. The outer surfaces of HHs/CNCs became more hydrophobic because of increasing crosslinking densities with extended exposure time in Fe³⁺

33

solutions. In comparison, the inner surface was highly hydrophilic due to the fluffy polymer mats at room temperature (Figure 20b).

In accordance with more crosslinking after longer treatment in Fe³⁺ solutions, HHs/CNCs with higher shell thicknesses became stronger (Figure 20c). HHs/CNCs with longer treatment in Fe³⁺ solutions (3 and 7 minutes) behaved similar as stiff hydrogels with larger deformations due to smaller internal volumes. In comparison, HHs/CNCs with immersion in Fe³⁺ solution for 1 minute containing larger internal volume was softer and too brittle to bear compression.

Figure 20. Regulation of microstructures and macrostructure of various HHs/CNCs. a) Shell thickness of HHs/CNCs with increasing immersion times in aqueous Fe³⁺ solutions. b) SEM images and water contact angles showing microstructures and wetabilities of freeze-dried HHs/CNCs with various treatment times in aqueous Fe³⁺ solutions. Insets are the photos of split freeze-dried HHs/CNCs with the scale bars of 2 cm. c) Compression tests of HHs/CNCs with various treatment times in aqueous Fe³⁺ solutions.

34

Figure 21. Shape programming of HHs/CNCs. Scale bar: 1 cm.

More importantly, the CNCs composite hydrogels exhibited self-healing property owing to the dynamic boronate ester bonds (Figure 21). In particular, the self-healed dynamic hydrogels

More importantly, the CNCs composite hydrogels exhibited self-healing property owing to the dynamic boronate ester bonds (Figure 21). In particular, the self-healed dynamic hydrogels

Im Dokument Functional Nanocomposite Hydrogels Based on Cellulose Nanocrystals (Seite 31-0)